PROJECT SUMMARY Root canals in permanent teeth have traditionally been treated by removing the necrotic tissue and replacing it with an artificial material. Regenerative endodontics has been proposed as an improved treatment option for these conditions. An imperative aspect in establishing the regeneration of vital dental pulp using tissue engineering methods, is an improved understanding of the conditions that are required to engineer the dental pulp vasculature. It has recently been demonstrated that a functional vasculature can be engineered by culturing endothelial cells and stromal cells from various sources in the correct microenvironmental conditions. However, the precise requirements specific to regenerating the pulp vasculature remain poorly understood. In the parent grant, we proposed to investigate key parameters that are important to regenerate the pulp vasculature, including matrix physical properties, composition and architecture. The proposed partner in this supplement (Dr. Christopher Chen) has developed and characterized a microfluidic bicellular model of the human vasculature which enables systematic studies of the interactions of endothelial and stromal cells with various exogenous agents. Recent data from Dr. Chen?s lab using this model has demonstrated that bacteria-derived inflammatory factors can significantly affect the function of engineered vascular capillaries, and the differentiation of stromal cells into pericytes, which are required to provide mural support to the engineered vessels. These recent findings are of central importance to the overarching goal of the parent award, since regeneration of dental pulp in adult teeth is typically performed in previously-infected teeth, where bacteria-derived inflammatory factors are retained within the dentinal tubules. Here we propose to combine recent developments resulting from the parent grant to engineer pulp-like tissue adjacent to dentinal tissue in a microfluidic device (tooth on-a-chip), with PI Chen?s biomimetic model of the human vasculature (vessel on-a-chip) to study the role of bacteria-derived inflammatory factors retained in dentinal tissue on the regeneration of the dental pulp vasculature. Ultimately this will allow us to engineer and characterize the first model of the vascularized dental pulp on-a-chip in healthy and infected conditions. We will first (aim 1.1) develop and characterize a microfluidic model of the vascularized dental pulp on-a-chip and will test the hydrogel elastic modulus can affect the differentiation of stromal cells into a pericyte- like lineage, thus leading to impaired vessel barrier function near the tooth. We will then (aim 1.2) study the effects of residual dentin infection on the formation of pericyte-supported vascular capillaries on-a-chip to test the premise that there is a detectable threshold of residual infection that impairs mature vasculature formation in pulp tissue.